Listeria monocytogenes, a Food-Borne Pathogen

Warnakulasuriya Inoka Priyadarshani Peiris Section of Food-Associated Pathogens Faculty of Veterinary Medicine and Animal Science

Swedish University of Agricultural Sciences Uppsala 2005

The present thesis is a partial fulfilment of the requirements for a Master of Science Degree in Veterinary Medicine for International Students at the Swedish University of Agricultural Sciences (SLU), in the field of Veterinary Public Health.

Warnakulasuriya Inoka Priyadarshani Peiris, Section of Food-associated Pathogens Faculty of Veterinary Medicine and Animal Science Swedish University of Agricultural Sciences (SLU) P. O. Box 7009, SE- 750 07 Uppsala, Sweden Print: SLU Service/Repro, Uppsala 2005

To my parents (W. R. I. Peiris and W. J. W. Peiris)

Abstract Peiris W. I. P. 2005. Listeria monocytogenes, a food-borne pathogen. Masters thesis. Section of Food-Associated pathogens, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences. ISSN 1403-2201. Listeria monocytogenes is a Gram positive, aerobic, facultative anaerobic and nonacid fast bacterium, which can cause the disease listeriosis in both human and animals. It is widely distributed thoroughout the environment and has been isolated from various plant and animal food products associated with listeriosis outbreaks. Contaminated ready-to-eat food products such as gravad and cold-smoked salmon and rainbow trout have been associated with human listeriosis in Sweden. The aim of this study was to analyse the occurrence and level of L. monocytogenes in gravad and cold-smoked salmon (Salmo salar) products packed under vacuum or modified atmosphere from retail outlets in Sweden. Isolated strains were characterized by serotyping and the diversity of the strains within and between producers were determined with PFGE (Pulsed-field gel electrophoresis). The characterized fish isolates were compared with previously characterized human strains. L. monocytogenes was isolated from 11 (three manufacturers) of 56 products analysed. This included gravad salmon products from three manufacturers and cold-smoked salmon from one manufacturer. The highest level of L. monocytogenes found was 1500 cfu/g from a cold-smoked salmon product but the level was low (90% of the inoculum, and spleen, which captures most of the remainder. Bacteria in the blood are rapidly cleared from the blood stream by resident tissue macrophages, kupffer cells, in the liver of experimentally infected mice (Conlan and North, 1991). Kupffer cells play a critical role in the development of immunity to infection (Gregory and Wing, 1990), and can promote the secretion of many factors including interlukin (IL-6, IL-1β), tumour necrosis factor (TNF)-α, and nitric oxide, which are capable of promoting inflammatory response and antimicrobial activity (Cousens and Wing, 2000). Bacteria multiply in the hepatocytes and spread cell-to-cell forming infectious foci. This type of spread prevents the bacteria to coming into contact with the humoral immunity. Hepatocytes respond to Listeria infection by releasing neutrophil chemotactants: neutrophil aggregation kills bacteria, lyses infected host cells, stimulates hepatocyte apoptosis and secretes cytokines that suppress intracellular replication in hepatocytes. This type of neutrophil-mediated defence mechanism can reduce >90% of bacterial growth in the liver during the first 24 hours of infection (Conlan and North, 1991). Listeria, which are not killed by neutrophils, are internalised by hepatocytes and undergo intracellular replication. Migrating Tcells rapidly eliminate infected hepatocytes. The antilisterial activity of T-cells is assisted by TNF-α and interferon (IFN)-γ (Gregory and Liu, 2000). If Listeria still survive due to inadequate immune response, rapid proliferation of L. monocytogenes in the liver hepatocytes may result in the release of bacteria into the blood stream resulting in infection of a wide range of host tissues.

L. monocytogenes in the brain Listeriosis associated with CNS (central nervous system) infection ranges from meningitis to meningoencephalitis. In ruminants, mainly the rhombencephalon is 16

affected: rhombencephalitis in ruminants is characterized by unilateral nerve paralysis leading to circling disease syndrome (J.C. Low and Donachie, 1997). However, the mechanism by which L. monocytogenes enters the CNS is poorly understood and various animal experiments provide evidence of different routes of infection. Intravenous inoculation occasionally results in CNS infection in sheep, whereas, intracarotid inoculation consistently results in encephalitis, suggesting the blood as the route of infection (Cordy et al., 1959; Jungherr et al., 1937; Olson et al., 1950). Neurological signs developed in mice after infection via intranasal and conjunctival routes, suggest the bacteria enter the brain by migration along the cranial nerves (Asahi et al., 1957). Experiments with rat dorsal root ganglia and hippocampal neurons, in culture (Dons et al., 1999) and clinical cases of ovine listerial encephalitis (Charlton, 1977) provide evidence of L. monocytogenes ability to infect axons and dorsal root ganglia nerve cell bodies and migrate in a retrograde as well as anterograde direction. Intra-axonal spread of L monocytogenes is demonstrated in mouse model (Otter and Blakmore, 1989). In an experiment, 6 out of 21 sheep developed neurological signs after injection of L. monocytogenes into dental pulp (Barlow and McGorum, 1985). In naturally infected humans and animals, Listeria have to cross the intestinal barrier and escape clearance mechanisms by the liver and spleen; therefore, invasion of the CNS is dependent on the concentration of bacteria in the blood and persistent bacteraemia (Berche, 1995; Blanot et al., 1997).

L. monocytogenes in the gravid uterus L. monocytogenes has a unique predilection for pregnant women which may result in serious outcomes such as abortion, still birth, or severe neonatal infections. Depressed cell-mediated immunity during gestation plays an important role in the development of listeriosis (Weinberg 1984). In an experimental murine model of pregnancy-associated listeriosis (Abram et al., 2002), impaired maternal immune response facilitates bacterial multiplication in the liver, placenta and foetal tissue. Listeriosis may occur at all stages of gestation but predominantly during the third trimester. L. monocytogenes gains access to the foetus by a haematogenous route by penetrating the placental barrier. In a guinea pig model, tropism of L. monocytogenes to the placenta and invasion, growth, and cell-to-cell spread of trophoblasts and trophoblast-derived cell lines has been demonstrated (Bakardjiev et al., 2004). The common clinical manifestations of infected pregnant women include, mild flulike illness with fever, headache, myalgia, arthralgia and malaise and occasionally gastrointestinal symptoms including abdominal pain and diarrhoea. There are few case reports of Listeria meningitis and meningoencephalitis in pregnant women (Boucher and Yonekura, 1984; McLauchlin et al., 1990).

17

USA USA Italy USA

Denmark

Canada Italy

Finland

New Zealand

Sweden

USA

Japan

1979 1989 1993 1994

1996

1996 1997

1999

2000

2001

2001

2001

38

16

>120

30

5

2 1566

3

23 2 18 45

No. of cases

Cheese

Turkey

Cheese

Imitation crab meat Maize and tuna salad Smoked rainbow trout Corned beef and ham

NR

NR Shrimp Rice salad Chocolate milk

Vehicle

1/2b

1/2a

1/2a

1/2

1/2a

1/2b 4b

4

4b 4b 1/2b 1/2b

Sero group

7 days (2-21 days) NR

NR

1.5 - 7 days

NR 3 days (1-7 days) NR

NR

NR NR NR NR

Illness duration

NR

27 / 32

NR, one L. m. joint abscess. NR

19 / 32

6/8

30/ 30

2/ 2

2/ 2 123 / 141

3/ 3

NR 0/2 NR 11 / 41

No.of stool samples (+)

NR

NR

1/1 1/ 40

1/1

No. of (+) blood samples for L.m. 18/20 2/2 2/4 3*/ 4

144 h

31 h (10-240 h) 25 h (6-49 h)

24 h (12-101 h)

12-18 h 24 h (6-51 h) 27 h

NR

NR 2 days 18 hours (11-60h) 20 hours (9-31 h)

Incubation period

Table 2. Reported listeriosis outbreaks mainly associated with gastrointestinal symptoms.

5 6 7 8 9 10 11 12

2.1×10 9 10 6 1.9×10 5 1.8×10 7 6.3×10 7 10 7 9.3x 108

1 2 3 4

Author

NR NR NR 1.2x109 8.8×108 NR

Cfu/g food

1. Ho et al., 1986; 2. Riedo et al., 1994; 3. Salamina et al., 1996; 4. Dalton et al., 1997; 5. Heitmann et a., 1997; 6. Farber et al., 2000; 7. Aureli et al., 2000; 8. Miettinen et al., 1999; 9. Sim et al., 2002; 10. Carrique-Mas et al.,2003; Danielsson-Tham et al., 2004; 11. Frye et al., 2002; 12. Makino et al., 2005. NR- Not reported, L. m. – Listeria monocytogenes. * - 3 additional patients found by surveillance not included in the picnic.

Place

Year

18

19

29

142

28

122

36

>300

New Zealand

Canada

USA

USA

Austria

Switzerl and

USA

U.K.

1980

1981

1983

1985

1986

19831987 19861987 19871989 Paté

4b

NR

NR

4b, 1/2a ½ b, 3b

NR

NR

NR

NR

NR

NR

35 or 31 days

NR

½a 4b

Raw milk, Vegetables Milk, soft cheese Ice cream or salami

NR

NR

NR

cfu/g of food

NR

NR

NR

Incubation period

4b

4b

4b

Pasteurised milk Cheese

4b

1b

Sero group

Coleslaw

Shellfish and raw fish.

Food associated

8

7

6

5

4

3

2

1

Author

42 immunosuppressed adults and 7 foetuses or newborn involved. Case fatality rate - 29% Pregnant women, neonates or foetuses predominantly affected. 10 neonatal and 20 foetal deaths. 98% had predisposing factors. Out of 28 patients, 24 were new born. 4 adults, only one showed compromised host defence. 65 occurred in newborn infants and pregnant women, 57 in adults. Out of the 57, 24 had underlying condition and mortality rate was 32%. 32 adults and 4 new born, one third of the patients had vomiting and diarrhoea. Thirteen of 15 patients had eaten paté within 3 weeks before onset of disease. Case fatality rate 32%. 31 materno-neonatal case patients. 9 foetal deaths. 2 healthy adults with CNS form of the disease.

34 perinatal listeriosis and 7 adults. 5 abortions, 4 stillbirths. No patient had evidence of any underlying immunosuppressive condition.

22 perinatal infections, 5 foetal deaths: common symptoms were flu and urinary tract symptoms.

Remarks

1993

France

38

Rillettes 21 days